Focal adhesion kinase (FAK) is a 125 kDa protein that colocalizes with integrins at focal adhesions upon cell adhesion to the extracellular matrix. FAK provides catalytic and scaffolding functions in integrin-mediated signaling events that control cell motility and survival. Multiple lines of evidence suggest that FAK may function in the pathology of human cancer and vascular disease and is therefore considered to be a potential target for drug development. The C-terminal focal adhesion targeting (FAT) domain of FAK mediates localization of FAK to discrete regions in the cell called focal adhesions and is important for FAK signaling since disruption of localization prevents the activation of FAK and phosphorylation of downstream substrates in response to integrin-dependent cell adhesion. Paxillin, a focal adhesion-associated adaptor protein that has been implicated in regulating cell motility, binds to the FAT domain and promotes FAK localization to focal adhesions. Moreover, phosphorylation of a strictly conserved tyrosine in the FAT domain modulates FAK localization, FAK signaling and focal adhesion turnover. We have previously solved NMR solution structures of the FAT domain in the presence and absence of a paxillin-derived peptide. We have also developed a novel methodology that integrates hydrogen exchange (HX) data into discrete molecular dynamics (DMD) simulations. The DMD/HX methodology was applied to the FAT domain of FAK and revealed the presence of a FAT intermediate state. The presence of this intermediate state is fully supported by experimental data leading us to propose that conformational dynamics of the FAT domain modulates paxillin binding and phosphorylation and therefore FAK function. The primary goal of this proposal is to investigate structural and dynamic features of the FAT domain that facilitate `switching'between phosphorylated and paxillin bound states of FAK using nuclear magnetic resonance (NMR) experiments and mutation studies combined with biochemical and biophysical approaches. Results from these studies are likely to shed light on how conformational dynamics of the FAT domain regulates FAK function and may provide information helpful for inhibition of FAK function by altering FAT domain ligand-binding and phosphorylation. Focal adhesion kinase (FAK) functions as both a scaffold protein and kinase that regulates a plethora of cellular processes such as cell proliferation, cell death and motility. Aberrant regulation of FAK can result in cancer and vascular disease. The focal adhesion targeting domain (FAT) is located at the C-terminus of the protein. Since the FAT domain regulates FAK function, the proposed investigation should provide information helpful for inhibiting FAK function by altering FAT domain ligand binding- binding and phosphorylation.